Compounds useful as photodynamic therapeutic agents

ABSTRACT

The present invention relates to compounds of the formula  
                 
or a salt, metal complex or hydrate or other solvate thereof, wherein: M is a chelating agent; 
     R 1 , R 2 , R 3 , R 4 , R 5  and R 6  are independently selected from the group consisting of: H; a substituted or unsubstituted, saturated or unsaturated, cyclic, moiety; a substituted or unsubstituted, saturated or unsaturated, heterocyclic moiety; or a substituted or unsubstituted, saturated or unsaturated, straight or branched chain alkyl or acyl moiety; and    R 2  and R 5  may also be independently a heavy atom or a water-solubilizing group. The present invention also relates to use of these compounds in the therapy in vivo or in vitro of a photosensitive target biological cell by irradiation, as well as methods of treating a photosensitive target biological cell in vivo or in vitro. Finally, the present invention relates to pharmaceutical compositions, comprising these compounds, in association with a pharmaceutically acceptable diluent or carrier.

The present invention relates to compounds useful as photodynarnictherapeutic agents and to pharmaceutical compositions containing saidcompounds. The present invention also relates to methods of photodynamictherapy, by administration of the said compounds.

Photodynamic therapy (PDT) is a non-invasive technique for the treatmentof a variety of solid tumour types by administering a photosensitisingcompound, followed by illumination of the tumour with light of awavelength absorbed by the compound, for example visible or near-visiblelight. The photosensitising compound is administered first to optimiseuptake of the photosensitising compound by the tissue to be treated. Atypical time lag between administration of the photosensitising compoundand subsequent illumination of the tissue would be 24-48 hours. PDT alsohas application in certain non-neoplastic diseases including age-relatedmacular degeneration, coronary heart disease and periodontal diseasescaused by overgrowth of pathogenic microflora around the teeth. Thetherapeutic strategy involves contacting a photosensitising compound oflow dark toxicity with a target area/tissue, which target area/tissue isin the body for in vivo therapies. The photosensitising compoundaccumulates preferentially to some extent within the target area/tissueto be treated, e.g., within a tumour. The target area/tissue is thenirradiated with low energy light through the body's therapeutic window,i.e. beyond the absorbance of body tissue, (650-850 nm), resulting inexcitation of the photosensitising compound. All other 2-5 things beingequal, the longer the wavelength of the illuminating light within thetherapeutic window, the greater the tissue penetration of light and,therefore, the greater the ability to treat deep seated tissues such asdeep seated tumours. The light-activated photosensitising compound canthen transfer its excited state energy to surrounding biological tissuethrough molecular oxygen, resulting in oxidative cellular damage leadingto cell death Via apoptosis and/or necrosis. After light treatment, thephotosensitiser is allowed to clear from the body. PDT can be viewed asa highly selective form of tissue treatment, provided that thephotosensitiser is non-toxic in the absence of light (i.e. has a lowdark toxicity), so that only the irradiated areas are affected.

Most known PDT compounds investigated to date are based oncyclic-tetrapyrrole macrocycles, from which it can be difficult togenerate a range of sequentially modified derivatives (M. Wainwright,Chem. Soc. Rev., 1996, 351).

At the present time, Photofrin®, a haematoporphyrin derivative, is themost commonly used clinically available PDT agent. It has been approvedfor use in the United States, Japan and Europe for the treatment ofoesophageal, lung, stomach, and cervical cancers (R. Bonnett, Chem. Soc.Rev., 1995, 24, 19 and T. J. Dougherty, C. J. Gomer, B. W. Henderson, G.Jori, D. Kessel, M. Korbelik, J. Moan, and Q. Peng, J. Natl. CancerInst. 1998, 90, 889). Although it is the most extensively usedanti-cancer PDT agent, it is widely recognised that it is far from beingan ideal drug for use in PDT (I. J. MacDonald and T. J. Dougherty, J.Porphyrins Phthalocyanines, 2001, 5, 105).

Other more recently approved agents are Foscan and Levulan, both ofwhich are porphyrin derivatives.

Despite its achievements to date, PDT is still in its developmentalstages, with a marked need to develop improved photosensitisingcompounds with better efficacy and side effect profiles. In order tofurther advance this novel form of treatment, it has become apparentthat the development of new PDT compounds, together with a more thoroughand integrated understanding of the multitude of targets/actions so farascribed to PDT agents, is needed.

The present invention alleviates the problems of the prior art byproviding synthesis, photophysical properties and in vitro cellularuptake evaluation of a new class of potential PDT agent, derived fromazadipyrromethenes whose tetraaryl derivatives 1 which were firstreported in 1940's but which, since then, have remained relativelyunstudied (M. A. T. Rogers, J. Chem. Soc., 1943, 596 and E. B. Knott, J.Chem. Soc., 1947, 1196).

A first aspect of the present invention, therefore, provides a compoundof the formula

in which M is a chelating agent; R¹, R², R³, R⁴, R⁵ and R⁶ can each,independently, be H; a substituted or unsubstituted, saturated orunsaturated, cyclic, preferably aryl, moiety; a substituted orunsubstituted, saturated or unsaturated, heterocyclic, preferablyheteroaryl, moiety; or a substituted or unsubstituted, saturated orunsaturated, straight or branched chain alkyl or acyl moiety and R² andR¹ can each, in addition and independently, be a heavy atom, preferablya halogen selected from At, I, Br or Cl, of which I or Br are mostpreferred, or a water-solubilizing group. The present invention alsoprovides salts, metal complexes or hydrates or other solvatesparticularly with lower, e.g., C₁-C₄, aliphatic alcohols of theaforementioned compounds.

R¹ and R⁶ (which may be the same or different, the same being preferred)are at the α-pyrrole positions; R² and R⁵ (which may be the same ordifferent, the same being preferred) are at the β3-pyrrole positions;and R³ and R⁴ (which may be the same or different, the same beingpreferred) are at the γ-pyrrole positions, all with respect to the Natom of each pyrrole ring.

Preferably, M is BX₂, in which each X is, independently, a halide. Mostpreferably, each halide is a fluoride. Alternatively, M is a metalselected, preferably, from Zn, Al, Si, Mg, Lu and Sn.

As used herein, the term “heavy atom” is intended to embrace atoms withan atomic weight greater than 15, preferably greater than 30, morepreferably greater than 35. Selenium is another example of a heavy atom.

As used herein, the term “cyclic” is intended to embrace substituted orunsubstituted, saturated or unsaturated, moieties containing one or morerings. If more than one ring is present, the rings may be fusedtogether. Suitable are substituted or unsubstituted steroids.

As used herein, the term “aryl”, which is included within the scope of“cyclic”, is intended to embrace substituted or unsubstituted,unsaturated, monocyclic or polycyclic (fused or separate) aromatichydrocarbon moieties. Preferred monocyclic aromatic moieties includephenyl, substituted phenyl moieties including, but not limited to,tolyl, xylyl, mesityl, cumenyl (isopropyl phenyl) and substitutedphenylene derivatives including, but not limited to, benzyl, benzhydryl,cinnamyl, phenethyl, styrl and trityl. Preferred fused polycyclicmoieties include substituted and unsubstituted naphthalene andanthracene moieties.

As used herein, the term “heterocyclic” is intended to embracesubstituted or unsubstituted, saturated or unsaturated, monocyclic orpolycyclic (fused or separate) heterocyclic moieties. Suitablenon-aromatic moieties are substituted or unsubstituted piperidine,dioxane, piperazine and pyrrolidine moieties.

As used herein, the term “heteroaryl”, which is included within thescope of “heterocyclic”, is intended to embrace substituted orunsubstituted, unsaturated, monocyclic or polycyclic (fused or separate)aromatic heterocyclic moieties. Preferred are substituted orunsubstituted pyridine, pyridazine, pyrimidine, pyrazine, purine, furan,pyrrole, benzofuran, indole and thiophene moieties.

As used herein, the term “aromatic” is intended to embrace a fullyunsaturated, substituted or unsubstituted, cyclic moiety.

As used herein, the term “alkyl” is intended to embrace substituted orunsubstituted, straight or branched chain, saturated or unsaturatedC₁₋₂₅ alkyl, alkenyl or alkynyl moieties. Preferred are alkyl moietiessuch as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,pentyl, isopentyl, hexyl, methylpentyl, isohexyl, heptyl, isoheptyl,octyl, isooctyl, nonyl, isononyl, decyl, isodecyl, undecyl, dodecyl,tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octodecyl,nonadecyl, icosyl, henicosyl, docosyl, tricosyl, tetracosyl andpentacosyl, all of which may be further substituted. Preferred alkenyland alkynyl moieties include vinyl, ethynyl, allyl, isopropenyl,propynyl, butenyl, butynyl, pentenyl, pentynyl, hexenyl, hexynyl,heptenyl, heptynyl, octenyl, octynyl, nonenyl, nonynyl, decenyl,decynyl, undecenyl, undecynyl, dodecenyl, dodecynyl, tridecenyl,tridecynyl, tetradecenyl, tetradecynyl, pentadecenyl, pentadecynyl,hexadecenyl, hexadecynyl, heptadecenyl, heptadecynyl, octadecenyl (oleicor elaidic), octadecynyl, nonadecenyl, nonadecynyl, icosenyl, icosynyl,henicosenyl, henicosynyl, docosenyl, docosynyl, tricosenyl, tricosynyl,tetracosenyl, tetracosynyl, pentacosenyl and pentacosynyl, all of whichmay be further substituted.

As used herein, the term “acyl” is intended to embrace alkyl-CO—moieties.

Advantageously, R¹ and/or R⁶ comprise, independently, a cyclic,preferably an aryl, moiety or a heterocyclic, preferably a heteroaryl,moiety (of the latter of which thiophene, furan or pyrrole moieties arepreferred). These moieties may be substituted or unsubstituted. Arylmoieties are more preferred, with monocyclic aryl moieties, and inparticular phenyl, being most preferred. R¹ and/or R⁶ preferably alsocontain an electron-donating substituent to maximise extinctioncoefficients and to shift the maximum wavelength of absorption beyond650 nm. Alkoxy (with a C₁₋₂₅ alkyl, preferably C₁₋₁₀ alkyl, and morepreferably C₁₋₄ alkyl group), most preferably methoxy, is a preferredelectron-donating substituent. Alternatively, R¹ and/or R⁶ may comprise,as an electron-donating substituent, a substituted or unsubstituted,saturated or unsaturated, straight or branched chain alkyl moiety (whichhas from 1 to 25, preferably 1 to 10 and more preferably 1 to 4, carbonatoms).

Advantageously, R² and/or R⁵ would comprise as a moiety a heavy atom,such as a halide, more preferably chloride, bromide or iodide and mostpreferably bromide or iodide, to maximise population of the tripletstate of the compound due to the “heavy atom effect”. The heavy atomeffect results in more efficient population of the triplet excited stateof a photosensitizing compound. This can result, in turn, in a moreefficient generation of singlet oxygen. Alternatively, if R² and/or R⁵is an alkyl, cyclic or heterocyclic moiety, it may be substituted withone or more heavy atoms, for example, a halide, more preferablychloride, bromide or iodide and most preferably bromide or iodide.

Alternatively, R² and/or R⁵ would comprise a moiety or include as asubstituent a water-solubilizing group to enhance the solubility ofcompounds of the present invention in aqueous solution. Suitablewater-solubilizing groups include a moiety derived from sulfonic acids(—SO₃H), alcohols (—OH), carboxylic acids (—COOH), amines (—NR₂, —N⁺R₃),amides (—NHCOR, —CONHR), tetrazoles (—CN₄R), sulphonamides (—NHSO₂R,—SO₂NHR) in which R is hydrogen or a substituted or unsubstituted,straight or branched chain alkyl moiety (which has from 1 to 25,preferably 1-10 and more preferably 1-4, carbon atoms).

R³ and/or R⁴ preferably comprise, independently, a cyclic, preferably anaryl, moiety or a heterocyclic, preferably a heteroaryl, moiety. Thesemoieties may be substituted or unsubstituted. Aryl moieties are morepreferred, with monocyclic aryl moieties, and in particular phenyl,being most preferred.

R³ and/or R⁴ may be substituted with one or more heavy atoms, forexample, a halide.

Advantageously R 3 and/or R⁴ comprise a moiety, or include substituents,that would maximise localisation of the compound in the tissue to betreated and optimise lipophilicity of the compound. Suitablesubstituents for alkyl; cyclic, preferably aryl; or heterocyclic,preferably heteroaryl, moieties to optimise lipophilicity include, butare not limited to, moieties derived from carboxylic acids (—COOH),sulfonic acids (—SO₃H), phenols (—OH), alcohols (—OH), amines (—NR₂,—N⁺R₃), amides (—NHCOR, —CONHR), tetrazoles (—CN₄R), sulphonamides(—NHSO₂R, —SO₂NHR) and esters (—COOR), in which R is a substituted orunsubstituted, straight or branched chain alkyl moiety (which has from 1to 25, preferably 1-10 and more preferably 1-4, carbon atoms).

Suitable substituents for alkyl, cyclic or heterocyclic moieties or,alternatively, suitable alkyl, cyclic or heterocyclic moieties toimprove localisation within the tissue to be treated, for example, thecancerous region include, but are not limited to, certain carbohydratesincluding β-D-galactose known to play a role in tumour cell recognition(C. Kieda, & Monsigny, M. (1986). “Involvement of membrane sugarreceptors and membrane glycoconjugates in the adhesion of 3LLsubpopulations to cultured pulmonary cells.” Invasion Metastasis, 6,347-366); certain tripeptide sequences including Arg-Gly-Asp andAsn-Gly-Arg known for their utility in targeting doxorubicin to newblood vessels within tumours (Barinaga, M. (1998) “Peplide-guided cancerdrugs show promise in mice” Science, 279, 323-324 and Arap, W.,Pasqualini, R., and Ruoslahti, E. (1998) “Cancer treatment by targeteddrug delivery to tumor vasculature in a mouse model” Science, 279,377-380); and certain steroids including 17β-oestradiol which mayincrease targeting of oestrogen receptor-positive breast cancer cells(Ferguson, A. T., Lapidus, R. G., and Davidson, N. E. (1998) “Theregulation of estrogen receptor expression and function in human breastcancer” Cancer Treat. Res., 94, 255-278).

Preferably, the compounds of the present invention have an extinctioncoefficient of greater than 30,000 M⁻¹ cm⁻¹, more preferably greaterthan 50,000 M⁻¹ cm⁻¹, even more preferably greater than 70,000 M⁻¹ cm⁻¹,and a maximum absorbance at greater than 640 nm, preferably greater than650 nm as measured in water: Cremophor solution (1100: (v/v)).Advantageously, the compounds of the present invention are, in vivo,localised within the cytoplasm, but not the nucleus, of the cells of thetarget tissue/area to be treated.

The compounds of the present invention, as non-porphyrin sensitisers,are a good starting point as they are amenable to modification of thephenyl rings around the periphery of the molecule to optimise theirtherapeutic properties. As a result of their ease of synthesis andpurification, arrays of compounds with systematic structural variationcan be generated to optimise the desired chemical, photophysical andbiological properties of the photosensitising compounds of theinvention.

In a second aspect, the invention provides a pharmaceutical composition,comprising a compound of the first aspect of the present invention inassociation with a pharmaceutically acceptable diluent or carrier.

In a third aspect, the invention provides a method of treating aphotosensitive target biological cell in vivo or in vitro, the methodcomprising contacting the target biological cell with an effectiveamount of a compound of the first aspect of the invention or with aneffective amount of a pharmaceutical composition of the second aspect ofthe present invention and then subjecting the photosensitive targetbiological cell with light absorbed by the said photosensitive cell, forexample light at a wavelength of greater than 570 nm, preferably greaterthan 600 nm, still more preferably greater than 650 nm.

In a fourth aspect, the invention provides use of a compound of thefirst aspect of the invention, preferably in association with apharmaceutically acceptable diluent or carrier, in the preparation of amedicament of use in the therapy in vivo or in vitro of a photosensitivetarget biological cell by irradiation.

In further aspects, the invention provides: a method of photodynamictherapy, comprising administering a compound of the first aspect of theinvention, preferably in association with a pharmaceutically acceptablediluent or carrier; and the use of a compound of the first aspect of theinvention, preferably in association with a pharmaceutically acceptablecarrier or diluent, in the manufacture of a medicament for the treatmentof tumours in association with light, preferably of a wavelength ofgreater than 570 nm, more preferably greater than 600 nm, still morepreferably greater than 650 nm.

These compositions are useful for sensitising a target biologicalsubstrate, for example, a tumour cell or other target, for example, anabnormal cell to destruction by irradiation using visible ornear-visible light.

Typical indications, known in the prior art, include destruction oftumour tissue in solid tumours; dissolution of plaques in blood vessels;treatment of topical conditions such as acne, athletes foot, warts,papilloma, psoriasis and treatment of biological products, such as bloodfor transfusion, for infectious agents.

The compositions are formulated in pharmaceutical compositions foradministration to the human or animal subject or applied to an in vitrotarget. The compositions can be administered systemically, in particularby injection, or can be used topically.

Injection may be intravenous, subcutaneous, intramuscular orintraperitoneal, injectable compositions can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Suitable excipients are, for example, water, saline,dextrose, glycerol and the like. Such compositions may also containminor amounts of non-toxic auxiliary substances such as wetting oremulsifying agents, pH buffers and the like.

Systemic administration can be achieved, alternatively, throughimplantation of a slow release or a sustained release system, forexample by suppository ororally, if so formulated.

If the treatment is to he localised, such as for the treatment ofsuperficial tumours or skin disorders, the compositions can be topicallyadministered using standard topical compositions such as lotions,suspensions or pastes.

The invention will now be further described, with reference to thefollowing non-limiting examples:—

Assay Methods

Cell Culture

MRC5 and HeLa cell cultures were maintained in MEM (minimum essentialmedium) containing 10% (v/v) foetal calf serum (FCS), 1% (v/v)non-essential amino acids, 2 μg/ml Fungizone (Trade Mark) (amphotericinB), 50 μg/ml penicillin, 50 μg/ml streptomycin, 20 mM HEPES and 1% (v/v)L-Glutamine. Cells were passaged at least twice before use in thecytotoxicity assays.

Cytotoxicity Studies (Haematoporphyrin)

Stock haematoporphyrin water-dimethyl sulphoxide (DMSO) (100:1 (v/v))solutions were diluted with MEM containing 10% FCS. Cells were seeded at5,000 cells/well in 96-well plates and incubated for 24 hr at 37° C. The24 hour incubation period was chosen having regard to the slower uptakeof haernatoporphyrin in the absence of Cremophor, when compared to thecompounds of the present invention in the presence of Cremophor (seebelow). Cells were then incubated with haematoporphyrin in the dark for24 hr at 37° C. The haematoporphyrin laden culture medium was thenremoved by filtration, the cells were washed with PBS and fresh culturemedium was added to each well. A 500W light source, passed through a redglass and water filter barrier, (to ensure cells are irradiated withlight of wavelength greater than 570 nm) was used to irradiate theplates for both 15 and 30 min. Following irradiation, the cells wereincubated for a further 48 hr at 37° C. before being assessed for cellsurvival. The dark toxicity of haematoporphyrin was also assessed ineach experiment to show that any measured cell death was due to lightillumination itself.

Cytotoxicity Studies (Compounds of the Invention)

Stock 2a or 2b water-Cremophor EL (Trade Mark —CAS 61791-12-6) (1:250(v/v)) solutions were diluted with MEM containing 10% FCS. Cells wereseeded at 5,000 cells/well in 96-well plates and incubated for 24 hr at37° C. Cells were then incubated with 2a or 2b in the dark for 3 hr at37° C. The 3 hour incubation period was chosen to reflect the fastercellular uptake of 2a or 2b in the presence of Cremophor, when comparedto the cellular uptake of haematoporphyrins in the absence of Cremophor.The 2a or 2b laden culture medium was then removed by filtration, thecells were washed with PBS and fresh culture medium was added to eachwell. The plates were then irradiated using a 500W light source passedthrough a red glass and water filter barrier, (thereby ensuring cellsare irradiated with light of wavelength greater than 570 nm) for both 15and 30 min.

Following irradiation, the cells were incubated for a further 48 hr at37° C. before being assessed for cell survival. The dark toxicity ofcompounds of the present invention was also assessed in each experiment.

Measurement of Cell Viability

Cell viability was estimated using the standard MTT (microtitertetrazolium) assay. This assay measures mitochondrial dehydrogenaseactivity and is based on the reduction of a soluble yellow tetrazoliumsalt to a blue, insoluble MTT formazan product by this enzyme. Thesubsequent colour change produced by viable cells was quantified using aplate reader (VICTOR² 1420 multilabel HTS counter, Wallac). As mentionedabove under the headings “Cytotoxicity”, the cells are incubated at 37°C. for 48 hours, following which the MTT solution was added to the cellsat a final concentration of 0.5 mg/ml and incubated for 3 hr at 37° C.The MTT solution was then removed by filtration and 100 μl DMSO wasadded to each well in order to lyse the cells and release the formazandye. The plates were read 1 hr later at 540 nm.

Preparation of Cells for Microscopy

Cells were seeded at a density of 30,000 cells/well in chamber slidesand allowed to adhere for 24 hr. 2a (10⁻⁵ M) was then added to eachchamber and incubated at 37° C. in the dark for either 1 hr or 3 hrs at37° C.—a one hour incubation was used for FIGS. 4 and 5. The medium wasremoved and the cells were washed 4 times with drug-free (2a free)medium. The cells were then fixed with 3.7% (v/v) formaldehyde for 15min at 37° C. The fixative solution was removed and the cells werewashed twice with sterile PBS. DAPI (4′, 6-diamidino-2-phenylindole)nuclear stain (1/1000) was then incubated with the cells for 10 min at37° C., following which the cells were washed twice with PBS. Cells werethen mounted in Vectashield (Trade Mark) mounting medium.

Fluorescence Microscopy

The cells were viewed using an Axio Zeiss fluorescent microscope. Thecells were examined at the different time points using two differentfilters. The rhodamine filter, which is specific for the wavelengthregion in which 2a fluoresces, was used to visualise 2a. The DAPI filterwas employed to examine the nuclei of the cells.

Confocal Microscopy

The cells were viewed using a Leica TCSSL Confocal Laser Scanningmicroscope (the LSM510 META Confocal Microscope was used to visualiseDAPI and 2a simultaneously).

Fluorescence Microscopy: Uptake Study

Cells were seeded at a density of 30,000 cells/well in chamber slidesand allowed to adhere for 24 hr. A compound of the present invention(10⁻⁵ M) was then added to each chamber and incubated at 37° C. in thedark over time, for example, 15 min, 30 min, 1 hr, 3 hr and 6 hr. Themedium was removed after the specified time and the cells were washed 4times with medium free of that compound of the invention. The cells werethen fixed with 3.7% (v/v) formaldehyde for 15 min at 37° C. Thefixative solution was removed and the cells were washed twice withsterile PBS. DAPI nuclear stain (1/1000) was then incubated with thecells for 10 is min at 37° C., following which the cells were washedtwice with PBS. Cells were then mounted in Vectashield (Trade Mark)mounting medium.

Fluorescence Microscopy: Efflux Study

Cells were seeded at a density of 30,000 cells/well in chamber slidesand allowed to adhere for 24 hr. A compound of the present invention(10⁻⁵ M) was then added to each chamber and incubated at 37° C. in thedark for 3 hrs. The same procedure was used as for the uptake studies,but for the efflux studies, the cells were treated with fixativesolution at specified times after removal of the drug, for example, 5min, 15 min, 30 min, 1 hr, 2 hr, 3 hr, 6 hr and 24 hr. The slides werethen viewed using an Axio Zeiss fluorescent microscope. The cells wereexamined at the different time points using two different filters. Therhodamine filter was used to visualise the compound of the presentinvention. Due to its inherent fluorescent properties, the compounds ofthe present invention fluoresce red when viewed under the rhodaminefilter. The DAPI filter was employed to look at the nuclei of the cells,which fluoresced blue due to treatment of DAPI, a nuclear stain.

LabWorks (Bioimaging Systems) was used to calculate the averagefluorescence intensity per cell for each of the different time pointsused in the uptake and efflux studies for the compounds of the presentinvention. For each time point, 5 fields of view were examined and eachuptake/efflux experiment was performed in duplicate. The DAPI filter wasused to accurately count the number of cells in each field of view andthe rhodamine filter was used to quantify the fluorescence of thecompounds of the present invention.

Data Analysis

Prism (Trade Mark) (Bioimaging Systems) was used to graph the dataobtained from the MTT assays and the uptake/efflux experiments. Thisprogramme allows non-linear regression analysis and the generation ofsigmoidal dose response curves. Prism (Trade Mark) also automaticallycalculates EC₅₀ values.

EXAMPLE 1

Referring to the accompanying reaction scheme, synthesis of 1 wasrepeated using the reported three step literature procedure of Rogers(1943). In order to make the chromophore more rigid, i.e., morestructurally constrained and to limit radiationless transitions, so itwould have the potential to act as a PDT agent, we converted it into itsBF₂ chelate 2 (72-83% yield) by reaction at room temperature for 16hours with boron trifluoride diethyl etherate, diisopropylamine (DIEA)in CH₂Cl₂. As the introduction of a heavy atom into a chromophore isgenerally accepted to facilitate enhancement of triplet state population(a requirement for singlet oxygen generation), we brominated at roomtemperature for 2 hours the free β-position of both pyrrole rings of 1with molecular bromine in toluene or benzene giving 3 in high yields(85-90%). Conversion of 3 into its BF₂ chelate 4 was readily achievedusing the same conditions as for 1 (see top part of Reaction Scheme—FIG.1)(71-78%). It will be appreciated that other compounds of the presentinvention can be similarly prepared, by use of the appropriatelysubstituted azadipyrromethene, in place of compound 1.

Both 2 and 4 are metallic brown solids and have good solubility inorganic solvents such as chloroform, toluene or THF (tetrahydrofuran)and were fully characterised by ¹H, ¹³C NMR and HRMS (high resolutionmass spectroscopy).

Compound 2a denotes Compound 2 of the Reaction Scheme, where Ar isPhenyl, whilst Compound 2b denotes Compound 2 of the Reaction Schemewhere Ar is paramethoxyphenyl. Similarly, Compounds 4a and 4b denote Aras phenyl or as paramethoxyphenyl, respectively.

Compound 2a:

¹H NMR (CDCl₃): 7.03 (2H, s), 7.40-7.53 (12H, m), 8.0-8.1 (8H, m).

¹³C NMR (CDCl₃): 119.3, 128.8, 128.8, 129.6, 129.7, 129.8, 129.9, 131.1,131.8, 132.5, 143.6.

EI-HRMS: 497.1868.

Compound 2b:

¹H NMR (CDCl₃): 3.85 (6H, s), 7.02 (6H, m), 7.45 (6H, m) 8.06 (8H, m).

¹³CNMR(CDCl₃): δ5.66, 114.51, 118.91, 124.42, 128.78, 129.45, 129.53,131.83, 131.89, 131.95, 132.76, 143.40, 162.20.

EI-HRMS: 557.2085.

Compound 4a:

¹H NMR (CDCl₃): 7.41-7.50 (12H, m), 7.70-7.73 (4H, m), 7.84-7.89 (4H, m)

¹³C NMR (CDCl₃): 109.8, 126.9, 127.0, 128.4, 128.6, 129.3, 129.5, 129.7,129.8, 141.9, 143.3, 157.5.

EI-HRMS: 653.0076.

Compound 4b:

¹H NMR (CDCl₃): 3.85 (6H, s), 6.9 (4H, d), 7.40-7.46 (6H, m), 7.75 (4H,d), 7.84-7.87 (4H, m).

³C NMR (CDCl₃): δ5.5, 110.2, 113.8, 122.0, 128.2, 129.7, 130.9, 131.0,132.7, 142.2, 144.0, 157.1, 161.7.

EI-HRMS: 713.0275.

EXAMPLE 2

A study of the spectroscopic properties of 2a and 4a in chloroformdemonstrated that they have a relatively sharp absorption band of650-660 nm of high molar extinction coefficients ˜80,000, with a fullwidth at half maximum (fwhm) of ˜50 nm. Introduction of an electrondonating methoxy group onto the phenyl rings adjacent to the pyrrolenitrogen resulted in an increase in extinction coefficient and asignificant bathochromic shift of the absorption bands for 2b and 4b at688 and 679 nm, respectively (Table 1, FIG. 2). The absorption bands ofeach photosensitiser are relatively insensitive to solvent changes withsolutions in water/Cremophor resulting in a further bathochromic shiftof ˜10 nm (Table 1). Excitation of chloroform solutions of the 2a and 4aat 635 nm gave a fluorescence band at 672 and 673 nm respectively (FIG.2, Table 2). The fluorescence quantum yield of 2a was 0.34 and, as wouldbe expected, is significantly reduced for 4a (0.012) due to the internalheavy atom effect (Table 2). Similarly, 2b had a fluorescence quantumyield of 0.36, while 4b was 0.10. The reduction in fluorescence quantumyield in the series would imply more efficient population of the tripletexcited state which would benefit singlet oxygen production. The abilityof 2 and 4 to produce singlet oxygen would be a prerequisite to thembeing potential PDT agents. TABLE 1 Spectroscopic absorbance propertiesof 2 and 4^(a) λmax^(b) fwhm^(b) ε^(b) λmax^(c) fwhm^(c) Compound (nm)(nm) (M⁻¹cm⁻¹) (nm) (nm) 2a 650 49 79,000 658 53 2b 688 55 85,000 696 574a 650 47 79,000 651 57 4b 679 57 75,000 685 86^(a)Room temperature.^(b)CHCl₃.^(c)Water/Cremophor. (100:1)

TABLE 2 Extinction coefficients and fluorescence quantum yields (Φ_(f))of 2 and 4^(a) Compound λ em (nm)^(b) Φ_(f) ^(c) λem (nm)^(d) 2a 6720.34 683 2b 715 0.36 727 4a 673 0.012 679 4b 714 0.10 719^(a)Room temperature.^(b)CHCl₃.^(c)Relative to magnesium tetra-tert-butylphthalocyanine in CHCl₃ (Φ_(f)= 0.84).⁷^(d)Water/Cremophor. (100:1)

Single crystal X-ray structure determination of 2b demonstrated theconjugated nature of the chromophore with similar bond lengths in bothpyrrole rings and is further confirmation of its molecular structure(FIG. 3).

EXAMPLE 3

Compound 5

Compounds 5a and 5b denote compounds of the present invention, in whichAr is phenyl or paramethoxyphenyl, respectively, and in which, in eachcase, the phenyl group, on each ring, furthest from the pyrrole N isreplaced with a parabromophenyl group.

This positions a heavy atom (if required) on the phenyl rings allowingR¹ and R⁶ incorporate an electron donating group e.g. para-OCH₃ (ifrequired) and the β-pyrrole substituent (R², R⁵) could be eitherunsubstituted or contain a group which imparts another advantage such asenhanced water solubility (if required).

The method of synthesis is the same as described in the original papersreferenced above and as outlined schematically below:—

The BF₃ chelate is prepared according to the bottom part of the reactionscheme of FIG. 1.

Compound 5a:

¹H NMR: 7.0 (2H, s), 7.47-7.61 (6H, m), 7.61 (4H, d), 7.90 (4H, d),8.01-8.05 (4H, m). λmax (CHCl₃): 658 nm. Extinction Coefficient 74,000M⁻¹ cm⁻¹. λλmax (H₂O-Cremophor): 667 nm. λem (CHCl₃): 680 nm.

EI-HRMS: 653.0081.

EXAMPLE 4

Cancer cellular uptake of a photosensitiser is a prerequisite for it toact as a PDT agent. Delivery of our proposed PDT agents requiredformulation of the sensitisers in order to impart water solubility.Water/Cremophor solutions of 2a (10⁻⁵ M) were added to HeLa cancer celllines and incubated for 5, 15, 60 and 120 mins, washed with water andexamined with fluorescent microscopy. Exploiting the inherentfluorescent properties of 2a, efficient uptake and cytosoliclocalisation of 2a was observed with maximum uptake after 300 minutes(FIG. 4). Dual staining of the nucleus of the cells with4′,6-diamidino-2-phenylindole (DAPI) prior to treatment with 2a gavegood contrast imaging and confirmed localisation of 2a primarily at theendoplasmic reticulum and not in the nucleus (FIG. 5).

EXAMPLE 5

Using the assay method set out above under the heading “FluorescenceMicroscopy”, the uptake of 2a was examined for HeLa and MRC5 cancer celllines and the data are illustrated in FIGS. 6 a and 6 b, respectively.Thus, for the Hela cells, a time-dependent uptake is illustrated with amaximum reached after 300 minutes whilst, for MRC5 cells, atime-dependent uptake is also shown, with a maximum reached after 200minutes.

EXAMPLE 6

Using the assay method set out above under the heading “FluorescenceMicroscopy”, the efflux of 2a was examined from Hela cells and the dataare illustrated in FIG. 7. The efflux is time-dependent, being completein 1000 minutes.

These efflux data suggest that the compounds of the present inventionare not retained in non-irradiated cells, post-treatment.

EXAMPLE 7

Preliminary light toxicity assays were carried out as follows:

HeLa cancer calls were exposed to 2a in varying concentrations for 24hours. 2a laden medium was removed and replaced with fresh medium. Cellswere irradiated at constant temperature of 37° C. for 15 minutes withlight from a 100 W or 500W halogen lamp passed through a red glass andwater filter barrier, thereby ensuring cells are irradiated with lightof more than 570 nm. Irradiated cells were incubated for a further 24hrs at 37° C. MT cell viability assay was performed.

A typical dose response curve is shown in FIG. 8. This demonstrates anEC-50 of 1×10⁻⁶ M using the 500W light source. The poorer response forthe 100 W light source demonstrates that varying the quantity of lightactivation has a direct effect on drug efficacy. The dark toxicityeffect may be caused by the micellar delivery vehicle itself.

EXAMPLE 8

Light toxicity assays were also carried out as described above under“Assay methods” in HeLa and MRC5 cells for haematoporphyrin (FIGS. 9 and10), 2a (FIGS. 11 and 12) and 2b (FIGS. 13 and 14).

Prior art haematoporphyrin was assessed with HeLa cancer cells. Exposureto 0 J/cm² light (no light) gave an EC50 value of 7.7×10⁻⁵M. Exposure to8 J/cm² light (15 mins from a 500W light source) or exposure to 16 J/cm²light (30 mins from a 500W light source) gave EC50 values of 1.5×10⁻⁵Mand of 1.6×10⁻⁵M, respectively.

Prior art haematoporphyrin was also assessed with MRC5 cancer cells.Exposure to 0 J/cm² light (no light) gave an EC50 value of 1.4×10⁻³M.Exposure to 8 J/cm² light (15 mins from a 500W light source) or to 16J/cm² light (30 mins from a 500W light source) gave EC50 values of4.2×10⁻⁵M or 2.9×10⁻⁵M, respectively.

Compound 2a was assessed with HeLa cells. Exposure to 0 J/cm² light (nolight) gave an EC50 value of 2.3×10⁻⁵M. Exposure to 8 J/km² light (15mins from a 500W light source) or to 16 J/cm² light (30 mins from a 500Wlight source) gave EC50 values of 4.5×10⁻⁶M and 1.3×10⁻⁶M, respectively.

Compound 2a was also assessed with MRC5 cancer cells. Exposure to 0J/cm² light (no light) gave an EC50 value of 3.5×10⁻⁶M. Exposure to 8J/cm² light (15 mins from a 500W light source) gave an EC50 value of2.0×10⁻⁶M, whilst exposure to 16 J/cm² light (30 mins from a 500W lightsource) gave an EC50 value of 4.6×10⁻⁷M.

Compound 2b was assessed with HeLa cancer cells. Exposure to 0 J/cm²light (no light) gave an EC50 value of 6.2×10⁻⁵M. Exposure to 8 J/cm²light (15 mins from a 500W light source) gave an EC50 value of5.3×10⁻⁵M. Exposure to 16 J/cm² light (30 mins from a 500W light source)gave an EC50 value of 2.5×10⁻⁵M.

Compound 2b was also assessed with MRC5 cancer cells. Exposure to 0J/cm² light (no light) gave an un-measurable EC50 value. Exposure to 8J/cm² light (15 mins from a 500W light source) gave an EC50 value of2.2×10⁻⁴M. Exposure to 16 J/cm² light (30 mins from a 500W light source)gave an EC50 value of 2.2×10⁻⁵M.

The EC50 data shows that both 2a and 2b are acting as PDT agents. 2a wasa significant improvement than the prior art compound haematoporphyrinand 2b was also improved in comparison to haematoporphyrin. Increasedlight doses from 8 J/cm² to 16 J/cm² give rise to more favourable EC-50values for both 2a and 2b thereby demonstrating that these compounds areacting as PDT agents as their effectiveness is dependant not only on theconcentration of compound administered to the cells but also thequantity of light energy delivered to the cells.

These toxicity data were confirmed by reference to FIG. 16 in contrastto FIG. 15.

FIG. 15 illustrates a control experiment which is a fluorescencemicroscope image of F-actin stained HeLa cells (with rhodaminephalloidine, red colour) 24 hours after treatment with Compound 2a andno exposure to light (0 min light dose). FIG. 15 illustrates suchF-Actin stained cells are live.

FIG. 16 confirms cell death following treatment with Compound 2a and 30minute 500W light dose, using fluorescence microscopy. Morespecifically, FIG. 16 is a fluorescence microscope image of F-actinstained HeLa cells (with rhodamine phalloidine, red colour) 24 hoursafter treatment with Compound 2a and 30 minute light dose.

EXAMPLE 9 Confocal Data

Referring to FIGS. 17-19, there are illustrated both diffuse andpunctuated cytoplasmic localisation of 2a. The diffuse spread issuggestive of mitochondrial localisation (see centre of cell of FIG.18). The punctuated spread is suggestive of localisation in organellesof size range 1-2 μm (see FIG. 19). These could be lysosomes which havea typical size range of 0.2-2 μm; peroxisomes which have a typical sizerange of 0.5-1.5 μm; or endosomes which have a typical size range of0.2-2 μm. It will be appreciated that localisation of thephotosensitising compounds of the invention in different subcellularsites may impact on how effective the photodynamic therapy may be.

1-14. (canceled)
 15. A compound of the formula

or a salt, metal complex or hydrate or other solvate thereof, wherein: Mis a chelating agent; R¹, R², R³, R⁴, R⁵ and R⁶ are independentlyselected from the group consisting of: H; a substituted orunsubstituted, saturated or unsaturated, cyclic, moiety; a substitutedor unsubstituted, saturated or unsaturated, heterocyclic moiety; or asubstituted or unsubstituted, saturated or unsaturated, straight orbranched chain alkyl or acyl moiety; and R² and R⁵ may, in addition, beindependently a heavy atom or a water-solubilizing group.
 16. A compoundas claimed in claim 15, wherein M is selected from the group comprising:BX₂, wherein each X is independently a halide; Zn; Al; Si; Mg; Lu; andSn.
 17. A compound as claimed in claim 16, wherein M is BF₂.
 18. Acompound as claimed in claim 15, wherein R¹, R₂, R³, R⁴, R⁵ and R⁶ areindependently selected from the group consisting of: H; a substituted orunsubstituted, saturated or unsaturated, aryl, moiety; and a substitutedor unsubstituted, saturated or unsaturated, heteroaryl moiety.
 19. Acompound as claimed in claim 15, wherein R¹ and R⁶ are optionallysubstituted phenyl.
 20. A compound as claimed in claim 19, wherein R¹and R⁶ contain an electron-donating substituent.
 21. A compound asclaimed in claim 20, wherein the electron-donating substituent isalkoxy.
 22. A compound as claimed in claim 15, wherein R² and R⁵ areselected from chlorine, bromine and iodine.
 23. A compound as claimed inclaim 15, wherein R² and R⁵ are selected from water-solubilizing groups.24. A compound as claimed in claim 15, wherein R³ and R⁴ are optionallysubstituted phenyl.
 25. A compound as claimed in claim 24, wherein R³and R⁴ are substituted by a halide selected from chlorine, bromine andiodine.
 26. A compound as claimed in claim 15 for use in a method ofphotodynamic therapy.
 27. A pharmaceutical composition comprising, inassociation with a pharmaceutically acceptable diluent or carrier, acompound of the formula

or a salt, metal complex or hydrate or other solvate thereof, wherein: Mis a chelating agent; R¹, R², R³, R⁴, R⁵ and R⁶ are independentlyselected from the group consisting of: H; a substituted orunsubstituted, saturated or unsaturated, cyclic, moiety; a substitutedor unsubstituted, saturated or unsaturated, heterocyclic moiety; or asubstituted or unsubstituted, saturated or unsaturated, straight orbranched chain alkyl or acyl moiety; and R² and R⁵ may, in addition, beindependently a heavy atom or a water-solubilizing group.
 28. Apharmaceutical composition as claimed in claim 27, wherein M is selectedfrom the group comprising: BX₂, wherein each X is independently ahalide; Zn; Al; Si; Mg; Lu; and Sn.
 29. A pharmaceutical composition asclaimed in claim 27, wherein R¹, R², R³, R⁴, R⁵ and R⁶ are independentlyselected from the group consisting of: H; a substituted orunsubstituted, saturated or unsaturated, aryl, moiety; and a substitutedor unsubstituted, saturated or unsaturated, heteroaryl moiety.
 30. Apharmaceutical composition as claimed in claim 27, wherein R² and R⁵ areselected from chlorine, bromine and iodine.
 31. A method of treating aphotosensitive target biological cell in vivo or in vitro, the methodcomprising the steps of contacting the target biological cell with aneffective amount of a compound of the formula

or a salt, metal complex or hydrate or other solvate thereof, wherein: Mis a chelating agent; R¹, R², R³, R⁴, R⁵ and R⁶ are independentlyselected from the group consisting of: H; a substituted orunsubstituted, saturated or unsaturated, cyclic, moiety; a substitutedor unsubstituted, saturated or unsaturated, heterocyclic moiety; or asubstituted or unsubstituted, saturated or unsaturated, straight orbranched chain alkyl or acyl moiety; and R² and R⁵ may, in addition, beindependently a heavy atom or a water-solubilizing group; and thensubjecting the photosensitive target biological cell with light absorbedby the said photosensitive cell.
 32. A method as claimed in claim 31,wherein M is selected from the group comprising: BX₂, wherein each X isindependently a halide; Zn; Al; Si; Mg; Lu; and Sn.
 33. A method asclaimed in claim 31, wherein R¹, R², R³, R⁴, R⁵ and R⁶ are independentlyselected from the group consisting of: H; a substituted orunsubstituted, saturated or unsaturated, aryl, moiety; and a substitutedor unsubstituted, saturated or unsaturated, heteroaryl moiety.
 34. Amethod as claimed in claim 31, wherein R² and R⁵ are selected fromchlorine, bromine and iodine.